氟对小鼠成釉细胞凋亡及c-myc表达的影响

目的观察氟化物作用下小鼠切牙成釉细胞凋亡及c-myc的表达,初步探讨c-myc在氟牙症发生中的作用。方法建立小鼠氟牙症模型,利用原位末端标记法(TUNEL)检测成釉细胞凋亡发生的情况,并利用免疫组织化学技术检测成釉细胞内c-myc的表达。结果TUNEL检测结果显示实验组小鼠切牙成釉细胞在各期凋亡数量均增多,与对照组比较有差异;原癌基因c-myc在实验组成釉细胞的分泌期表达增强,分泌前期表达较弱;实验组和对照组比较有显著性差异。结论氟作用下细胞因子c-myc的表达失调可促进成釉细胞凋亡,产生氟牙症病理改变。     

氟化物对大鼠切牙骨桥蛋白表达影响的实验研究

目的研究过量氟对牙硬组织发育过程中骨桥蛋白(OPN)时空表达的影响,从蛋白水平上进一步探讨氟牙症的发病机制。方法 20只Wistar大鼠随机分成两组:对照组(饮用蒸馏水)和实验组(100mg/LF-)。饲养8周后处死动物,通过免疫组织化学染色法观察并且比较OPN在对照组与实验组大鼠牙胚上皮中的表达,采用计算机图像分析系统对免疫组织化学染色阳性结果进行分析,采用SASv6.12统计软件对两组图像分析结果数值进行统计学t检验。结果对照组,各期成釉细胞排列均匀整齐,细胞形态正常,成牙本质细胞排列整齐,成栅栏状,牙本质均匀一致,转换期开始内釉上皮细胞和牙髓组织中OPN表达阳性,到成熟期时的阳性反应最明显;实验组,大鼠切牙成釉细胞由原有的高柱状变矮,细胞排列成多层,成牙本质细胞排列紊乱,釉基质形成混乱,大鼠牙胚上皮形成各期OPN表达则明显低于对照组。统计学t检验两组有显著性差异(P<0.01)。结论氟化物能抑制大鼠牙胚上皮OPN的表达。提示氟可能通过抑制OPN在牙胚发育过程中的表达,从而抑制牙胚上皮细胞(造釉细胞、成牙本质细胞、成牙骨质细胞)的增殖分化及随后的基质合成与分泌,导致氟斑牙的形成。     

镁硒联合对氟中毒小鼠切牙牙胚的影响

目的:观察镁硒联合预防氟牙症的效果及初步机理.方法:40只体质量18～24 g雄性小鼠,随机分为Ⅰ组(双蒸水、常规饲料)、Ⅱ组(饮用F-浓度为50 mg/L的双蒸水、常规饲料)、Ⅲ组(双蒸水、添加MgSO4·7H2O 162.5 mg/kg+Na2SeO3·5H2O 2 mg/kg饲料)、Ⅳ组(饮用F-浓度为50 mg/L的双蒸水、添加MgSO4·7H2O 162.5 mg/kg+Na2SeO3·5H2O 2 mg/kg饲料).每组10只.定期观察小鼠生长情况,42 d后处死,获取切牙标本,HE染色观察成釉细胞形态.结果:Ⅳ组分泌期成釉细胞形态较Ⅱ组有明显改善.结论:镁硒联合运用可拮抗过量氟对成釉细胞的损伤,对氟牙症有较好的预防作用.     

骨形态发生蛋白-13促进小鼠骨髓干细胞的软骨分化

目的:研究骨形态发生蛋白-13对小鼠骨髓基质干细胞向软骨分化过程的作用。方法:体外培养小鼠骨髓基质干细胞(MSCs),贴壁细胞传代,取第3代细胞,用骨形态发生蛋白-13溶液干预培养1,4,7,14 d取样检测。倒置相差显微镜观察细胞形态;RT-PCR,western-blot和免疫细胞化学法检测不同时期Ⅱ型胶原,SOX9,阿尔辛蓝(Alcian)染色蛋白多糖。结果:Ⅱ型胶原和SOX9之mRNA和蛋白的表达在第4天开始出现并不断升高;Alcian染色结果显示骨形态发生蛋白-13诱导细胞分泌蛋白多糖基质,可见细胞小结区域呈明显的异染性。结论:骨形态发生蛋白-13可以定向诱导小鼠骨髓干细胞的软骨分化。     

1,25-二羟维生素D3通过TGF-β1（/ Smad2/3）影响的ROS调节气道重塑 #br#

目的 探讨 1，25-二羟维生素 D3［1，25-（OH）2D3］通过转化生长因子-β1（TGF-β1）（/ Smad2/3）对活性氧 （ROS）的影响及调节气道重塑的分子机制。方法 选取健康雌性 Balb/c小鼠 24只，随机分为正常组（A组）、哮喘组 （B组）、1，25-（OH）2D3+哮喘组（C组）。B组和 C组于第 0、7、14天腹腔注射 0.5 mL致敏液（10 µg卵清蛋白与 2 mg铝制剂），之后用卵清蛋白雾化吸入，于第 21~27天每天雾化 1次，第 28~77天隔天雾化 1次制备小鼠支气管哮喘气道重 塑模型。C组在每次雾化激发前 30 min予以腹腔注射 100 ng 1，25-（OH）2D3，实验过程持续至少 77 d。收集小鼠肺组 织分别用于 HE染色、AB-PAS染色、Masson染色分析小鼠气道病理形态改变、气道黏液高分泌及气道重塑，运用计算 机图像分析系统评价各组气道重塑情况。用免疫荧光检测气道 ROS的表达，Western blot检测 TGF-β1、Smad2/3的表 达水平。结果 B组较 A组气道受损明显，可见大量炎性细胞浸润，黏液分泌增加，上皮下胶原沉积明显增多，与 B 组相比，C组病理形态损伤表现明显缓解，但仍较 A组加重；B组较 A组 ROS，TGF-β1、Smad2/3蛋白的表达水平明显 升高。与 B组相比，C组 ROS，TGF-β1、Smad2/3蛋白的表达降低，但仍较 A组升高（P＜0.01）。结论 1，25-（OH）2D3 可能通过抑制 TGF-β1（/ Smad2/3）的表达从而减少 ROS水平，达到调节小鼠气道重塑的作用。     

明胶海绵复合重组人骨形态发生蛋白-7植入物的体内异位成骨实验

目的：考察以明胶海绵为载体的重组人骨形态发生蛋白-7（rhBMP-7）在小鼠体内异位成骨能力,评价大肠杆菌表达的rhBMP-7生物学活性。方法：昆明种小鼠随机分为两组,将复合rh-BMP-7的明胶海绵植入小鼠肌间隙作为实验组,对照组植入明胶海绵,分别于术后1、2、3、4周取出埋植材料,HE染色进行组织学观察,测定蛋白含量、钙含量与碱性磷酸酶（ALP）活性。结果：复合rhBMP-7的明胶海绵组在术后2、3、4周有骨细胞类似细胞和钙化灶的形成,术后2、3、4周蛋白含量、ALP活性和钙含量均明显高于明胶海绵对照组。结论：明胶海绵复合rhBMP-7植入小鼠体内有较强的异位成骨能力,是良好的生物载体,可用于rhBMP-7体内活性检测。     

依达拉奉对帕金森病小鼠模型中脑细胞自噬相关蛋白表达的影响北大核心CSCD

目的研究依达拉奉对帕金森病(PD)小鼠模型中脑细胞自噬相关蛋白表达的影响。方法将42只C57BL/6雄性小鼠被随机分为对照组、模型组及实验组,各14只。模型组及实验组小鼠均腹腔注射25 mg·kg^(-1)1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)建立PD小鼠模型,对照组则腹腔注射等量生理盐水;建模成功后实验组腹腔注射3 mg·kg^(-1)依达拉奉,对照组及模型组则腹腔注射等量生理盐水,连续干预2周。以爬杆实验及悬挂实验对比小鼠行为学变化;用免疫组化法检测中脑黑质脑组织自噬相关蛋白表达情况。结果与对照组比较,模型组小鼠干预后1,2周爬杆时间均延长,悬挂实验评分降低(P<0.01);与模型组比较,实验组小鼠干预后1,2周爬杆时间均缩短,悬挂实验评分升高(P<0.01)。免疫组化显示,干预后2周,对照组、模型组及实验组小鼠中脑黑质脑组织Beclin1阳性细胞比例分别为(7.04±1.98)%,(27.05±2.01)%,(16.46±1.87)%;微管相关蛋白轻链3(LC3)阳性细胞比例分别为(5.41±1.67)%,(21.26±1.51)%,(14.86±1.49)%。与对照组比较,干预后1,2周模型组小鼠中脑黑质脑组织Beclin1及LC3阳性细胞比例明显增多(P<0.01);而实验组Beclin1及LC3阳性细胞比例较模型组明显降低(P<0.01)。结论依达拉奉可有效抑制PD小鼠中脑黑质脑组织细胞自噬相关蛋白Beclin1及LC3表达,改善PD小鼠异常行为。     

养血安胎方调节自噬治疗复发性自然流产小鼠的机制研究

目的 探讨养血安胎方调节自噬影响胎盘血管生成进而治疗复发性自然流产（RSA）的机制。方法 对妊娠小鼠分别使用雷帕霉素、3-甲基腺嘌呤及养血安胎方进行干预,收集小鼠胎盘,苏木素伊红（HE）染色、马松（Masson）染色观察妊娠小鼠的病理改变,免疫组化法测定增殖细胞核抗原（PCNA）、Fibrinogen及CD34蛋白的表达,Western blot法测定血管内皮生长因子（VEGF）、血管内皮细胞生长因子受体2(p-VEGFR2)、p62、Beclin1、LC3Ⅰ和LC3Ⅱ蛋白的表达,透射电镜观察胎盘的超微结构。结果 与正常妊娠组比较,雷帕霉素（Rapa）组小鼠取材时体重明显减轻,胚胎吸收率明显增加,HE染色、Masson染色及PCNA与Fibrinogen蛋白表达显示小鼠胎盘发育变差;透射电镜结果与p62、Beclin1和LC3Ⅱ/LC3Ⅰ蛋白表达显示小鼠胎盘自噬水平明显增加;CD34、VEGFA和p-VEGFR2蛋白表达显示胎盘血管生成异常;与Rapa组比较,Rapa+养血安胎组小鼠取材时体重明显增加,胚胎吸收率明显降低,HE染色、Masson染色及PCNA与Fibrinogen蛋白表...     

骨形态发生蛋白-2在糖尿病大鼠种植体周围的表达

目的:应用大鼠糖尿病模型,观察实验性2型糖尿病(T 2DM)大鼠种植体周围骨形态发生蛋白-2(BM P-2)的表达水平,探讨影响糖尿病种植体骨整合的分子生物学机制,试图为发现新的治疗糖尿病骨整合方法打下理论基础。方法:将48只大鼠均分为正常组和糖尿病组。糖尿病组按40 m g/kg腹腔内一次性注射枸橼酸钠链脲佐菌素溶液建立T 2DM模型。在胫骨近骺端种植纯钛种植体。种植后2,4,8周分批分次处死动物。采用不带种植体脱钙标本硬组织切片、脱钙标本切片常规苏木精-伊红染色(HE染色)和免疫组织化学法检测种植体周围骨组织中骨形态BM P-2的表达并做图像分析。结果:HE染色镜下观察糖尿病组骨形成滞后。正常组和糖尿病组的种植体周围骨组织BM P-2免疫组织化学灰度值两组间差异有显著性(P<0.05)。结论:糖尿病者的骨质疏松化倾向可能与糖尿病种植修复较正常者失败率高有关,糖尿病者BM P-2的减少可能是影响种植体骨整合的原因之一。     

百草枯中毒致小鼠肺纤维化模型的建立

目的探究百草枯中毒致小鼠肺纤维化模型的建立。方法无特定病原体级别(SPF)的BALB/c小鼠(6~8周龄,雄性,18~22 g)40只,分为A组(空白对照组)、B组(10 mg/kg百草枯单次染毒组)、C组(20 mg/kg百草枯单次染毒组)和D组(隔日1次百草枯染毒组,共3次),采用腹腔注射方式染毒观察至第21天取材,建立小鼠肺纤维化中毒模型。镜下观察组织HE染色变化和马松染色等纤维化改变和不同浓度的生存分析。结果20 mg/kg单次染毒组的小鼠HE染色和马松染色均显示纤维化改变,Ashcroft评分最高,肺组织上皮标志物和间质标志物的蛋白检测均显示有上皮间质改变。结论20 mg/kg腹腔注射单次给药至染毒后21 d的方法建立纤维化模型最好。     

Combined effect of fluoride and 2,3,7,8-tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin on mouse dental hard tissue formation in vitro

Fluoride interferes with enamel matrix secretion and mineralization and dentin mineralization. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), also impairs dental hard tissue formation and mineralization in vitro and in vivo. Our aim was to investigate in vitro whether the combined effect of sodium fluoride (NaF) and TCDD on dental hard tissue formation is potentiative. For this purpose, mandibular first and second molar tooth germs of E18 mouse embryos were cultured for 5–12 days with NaF and TCDD alone at various concentrations (2.5, 5, 10, 12.5, 15, and 20 μM and 5, 10, 12.5, and 15 nM, respectively) to determine the highest concentrations, which alone cause no or negligible effects. Morphological changes were studied from the whole tooth photographs and histological tissue sections. The concentrations found were 15 μM for NaF and 10 nM for TCDD. While at these concentrations, the effects of NaF and TCDD alone were barely detectable, the effect of simultaneous exposure on dentin and enamel formation was overt; mineralization of predentin to dentin and enamel matrix secretion and mineralization were impaired. Immunohistochemical analysis revealed that the combined exposure modified amelogenin expression by odontoblasts. Morphology of ameloblasts and the expression of amelogenin indicated that ameloblasts were still secretory. The results show that NaF and TCDD have potentiative, harmful effects on the formation of dental hard tissues. Since children can be exposed to subclinical levels of fluoride and dioxins during early childhood, coincidently with mineralization of the first permanent teeth, this finding may have clinical significance.     

Excessive fluoride impairs autophagy flux in ameloblasts which is prevented by the autophagy activator rapamycin

Excessive fluoride intake can cause dental fluorosis during teeth development and growth. However, the mechanisms underlying fluoride-induced enamel damage are still not fully elucidated. Previously, we observed fluoride-induced autophagy in ameloblasts, but the effects of fluoride on autophagy flux in ameloblasts remain unclear. Hence, this study aimed to clarify the effects of fluoride and rapamycin, an autophagy activator, on autophagy flux in ameloblasts. This in vitro study used the murine ameloblast-derived cell line LS8. Cells were treated with different concentrations of sodium fluoride (NaF) to evaluate NaF-induced cytotoxicity. Using transmission electron microscopy, we observed an increase in the number of autophagosomes with increasing fluoride concentrations. Western blot analyses showed increases in microtubule-associated protein 1 light chain 3 (LC3) and SQSTM1 (p62) expression after NaF treatment and an increase in LC3II expression after bafilomycin A1 administration. Together with changes in RFP-GFP-LC3 lentivirus expression, this demonstrated that fluoride impaired autophagy flux. Furthermore, we evaluated whether rapamycin can alleviate fluoride-induced cytotoxicity by restoring autophagy flux. Compared to the NaF-treated group, LS8 cells cotreated with NaF and rapamycin grew considerably better and had significantly decreased p62 expression. Taken together, these data suggest that fluoride-induced impaired autophagosome degradation may damage ameloblasts. This provides experimental in vitro evidence and an explanation for the observed NaF-induced toxicity of ameloblasts. Rapamycin probably alleviates this impairment by decreasing the expression of p62, thereby preventing autophagy defects.     

The dose-time effects of fluoride on the expression and DNA methylation level of the promoter region of BMP-2 and BMP-7 in rats

Skeletal fluorosis is a chronic metabolic bone disease caused by excessive exposed to fluoride. Recent studies have shown that fluoride causes abnormal bone metabolism through disrupting the expression of Bone Morphogenetic Proteins (BMPs). However, the relationship between fluoride and BMPs is not fully understood, and the mechanism of fluoride on BMPs expression is still unclear. This study investigated the dose-time effects of fluoride on BMP-2 and BMP-7 levels and DNA methylation status of the promoter regions of these two genes in peripheral blood of rats. Eighty Wistar male rats were randomly divided into four groups and treated for 1 month and 3 months with distilled water (control), 25 mg/L, 50 mg/L or 100 mg/L of sodium fluoride (NaF). Rats exposed to fluoride had higher protein expression of BMP-2 and BMP-7 in plasma at 1 month and 3 months. An increase in BMP-2 expression was also observed with an increase of fluoride exposure time. Significant hypomethylation was observed in 2 CpG sites (CpGs) of BMP-2 and 1 CpG site of BMP-7 promoter regions in the fluoride treatment groups. It concludes that fluoride has a dose-response effect on BMP-2 in fluorosis rats, and fluoride-induced hypomethylation of specific CpGs may play an essential role in the regulation of BMP-2 and BMP-7 expression in rats.     

糖尿病大鼠骨折愈合过程中 VEGF、BMP-2及BMP-7的动态观察

目的：动态观察糖尿病模型大鼠骨折愈合过程中的血管内皮细胞生长因子（ VEGF）、骨形态发生蛋白-2（BMP-2）、骨形态发生蛋白-7（BMP-7）的变化趋势,探索影响糖尿病骨折愈合的机制。方法选取Wester 雄性大鼠70只,采用随机数字表法分为糖尿病组[（腹腔注射链脲佐菌素（ STZ）造模]和对照组,每组35只。采用线锯锯断2组大鼠的左侧胫骨,采用外固定复位处理,观察2组大鼠术后第1、3、5、7周的血清及骨痂中VEGF、BMP-2、BMP-7的表达变化。结果糖尿病组大鼠术后第1周、术后第3周的VEGF、BMP-2、BMP-7的表达水平均显著低于对照组（ P＜0构．05）,术后第5、第7周2组大鼠骨痂组织中的VEGF、BMP-2、BMP-7的表达水平差异无统计学意义（ P ＞0．05）。糖尿病组与对照组大鼠的血清中VEGF、BMP-2、BMP-7在术后第1～7周差异无统计学意义（ P ＞0．05）,糖尿病大鼠术后第1、第3周BMP-7的表达水平显著低于对照组大鼠,差异有统计学意义（ P ＜0．05）。结论糖尿病大鼠血清、骨痂组织中BMP-7、BMP-2及VEGF表达减少可能是其延迟愈合的一个重要原因。     

Mechanism of toxic action of fluoride in dental fluorosis: whether trimeric G proteins participate in the disturbance of intracellular transport of secretory ameloblast exposed to fluoride

In enamel fluorosis model rats treated with sodium fluoride, secretory ameloblasts of incisor tooth germs exhibited disruption of intracellular trafficking. We examined whether heterotrimeric G proteins participated in the disruption of vesicular trafficking of the secretory ameloblast exposed to fluoride, using immunoblotting and pertussis toxin (IAP)-induced adenosyl diphosphate (ADP)-ribosylation for membrane fractions of the cell. Immunoblotting of crude membranes, post supernatants of the ameloblast, with anti-G_i3/o and anti-G_s antibodies showed that G_i3 or G_o proteins existed in the secretory ameloblast, but G_s protein did not. Immunoblotting of the subcellular membrane fractions indicated that the G_i3 or G_o proteins were located in the Golgi membrane, but were not in the rough endoplasmic reticulum (rER) membrane. Autoradiograph of IAP-induced ADP-ribosylation, however, showed the existence of IAP-sensitive G proteins both in rER and Golgi membranes. Fluoride treatment decreased the G proteins bound to both membranes. These findings indicate that different G proteins, both of which are IAP-sensitive, are present in the rER and Golgi apparatus, and suggest that these G proteins participate in the disturbance of intracellular transport of the secretory ameloblast exposed to fluoride. Received: 24 June 1998 / Accepted: 8 September 1998     

硒、钼、硼对氟中毒大鼠成釉细胞形态及PCNA、C-myc表达变化的影响

目的 观察硒、钼、硼对氟中毒大鼠成釉细胞形态及增殖细胞核抗原(PCNA),C-myc 基因表达变化的影,气方法大鼠分组均喂以含F-45 mg/L的蒸馏水及含不同浓度硒、钼、硼的饲料,建立微量元素拮抗氟中毒的大鼠模型.采用HE染色及免疫组化的方法观察氟对成釉细胞的影响,采用图像分析技术时免疫组化结果进行量化分析.结果 实...     

Influence of fluoride on secretory pathway of the secretory ameloblast in rat incisor tooth germs exposed to sodium fluoride

 Fluoride, which is an environmental toxicant, is a potent inducer of mottled enamel in humans and rats. To define the influence of fluoride on the secretory pathway in enamel fluorosis, mottled enamel was induced in the incisor tooth germs of rats by subcutaneous injections of sodium fluoride for 4 days, and then morphological and cytochemical changes of the secretory ameloblast were examined in the tooth germs with HRP-labeled lectin (Con A, GS-I, SBA and PNA) and En3 antibody labeling amelogenins. The accumulation of small vesicles on the route of the secretory pathway between the rER and the Golgi apparatus, disorder of Golgi stacks, and formation of abnormal large granules in distal cytoplasm were seen in the secretory ameloblast. Lectin staining patterns of the secretory ameloblast indicated the disturbance of the vesicular transport between the rER and the Golgi apparatus, and disorganization of the Golgi stack. Immunolabeling of the cell showed disruption of the sorting and fusion process on the secretory pathway. These results suggest that the fluoride disturbs the intracellular transport in the synthesis-secretory pathway of the ameloblast, and that this effect of fluoride on the synthesis-secretory pathway participates in the formation of enamel fluorosis. Received: 9 August 1995 / Accepted: 17 October 1995     

Kinetics of fluoride removal from plasma and bone of rats after chronic intake of fluoride

This study evaluated the kinetics of fluoride in plasma, femur surface and the whole femur of rats, after chronic exposure to different water fluoride levels was interrupted. Four groups of Wistar rats received drinking water containing 0, 5, 15 or 50 microg F/ml for 60 days (n = 50/group). The animals were euthanized immediately after exposure to fluoride or after 7, 30, 90 or 180 days (n = 10/subgroup). Plasma and femurs were collected. Fluoride on the femur surface, whole femur and plasma was analyzed with an electrode. Data were analyzed using ANOVA and Tukey's test (p< 0.05). The increase in plasma fluoride levels was significant only for the 50 microg F/ml group at 0 and 7 days. Regarding bone surface and whole bone, for most groups, significant increases in fluoride concentrations were observed with the increase in water fluoride concentrations at each time of euthanasia. For fluoride doses up to 15 microg F/ml, femur surface fluoride levels were reestablished 180 days after the exposure was discontinued, which was not valid for whole femur or for higher fluoride doses. We found a different kinetics of fluoride in plasma, femur surface and the whole femur of rats after chronic exposure to fluoride is interrupted.